Genetic engineering provides methods for the cloning of foreign genes, their cDNA sequences or portions thereof, and their introduction into bacterial host cells such as Escherichia coli. The level of production of a foreign protein in the host cell depends on appropriately arranged transcription and translation control sequences that permit the regulated expression of the desired proteins or peptides coded by the foreign genes, gene fragments, various gene fusions or mutants thereof.
Transcription (i.e., the process leading to the production of mRNA) is a key step in the expression of a gene. Transcription initiation occurs at specific promoters which regulate gene expression. Promoters are regulated negatively by repressors and positively by activator proteins. The initiation of transcription can be separated into several steps (Buc et al., Biochemistry (1985) 24:2712). In order to act effectively, promoters are made of three elements. A core sequence is recognized by RNA polymerase. This region is flanked by the USR, or the upstream region, and the DSR, or the downstream region. The USR was shown to bind specific activator proteins (Adhya et al., Gene (1993) 132:1). Two modes of gene activation are generally considered. DNA-bound activator makes direct contact with RNA polymerase and thereby facilitates the binding and isomerization of RNA polymerase. Alternately, the bound activator changes the structure of the promoter, thereby favoring transcription initiation.
In order to maintain the desired foreign gene and to express it in a bacterial host cell at a high level for protein purification, it is essential to place the gene under the control of a regulated promoter that permits turning off of gene expression during the first stage of fermentation in which the cell mass is increased, and turning it on at the second fermentation stage in which maximal gene expression and protein production is needed.
Production of proteins utilizing a number of promoters and ribosome binding sites has been previously described. For example, the use of the pL promoter for regulated gene expression has been the subject of several references (e.g., Bernard et al., Gene (1970) 5:59; Derom et al., Gene (1982) 17:45; Gheysen et al., Gene (1982) 17:55; Hedgpeth et al., Mol. Gen. Genet. (1978) 163:197; Remaut et al., Gene (1981) 15:81; and Deryneck et al., Nature (1980) 287). A number of ribosome binding sites have been employed in order to obtain a high level of protein synthesis, such as the phage .lambda. cII translation initiation region used for the production of cII (Oppenheim et al., J. Mol. Biol. (1982) 158:327; Shimatake et al., Nature (1981) 292:128). Similarly, production of human growth hormone (hGH) and bovine growth hormone (bGH) are described, inter alia, in U.S. Pat. No. 4,997,916.
One of the major problems in producing eukaryotic proteins in the bacterium E. coli is that the desired protein is often obtained in inactive aggregates termed inclusion bodies. The inclusion bodies can facilitate protein purification provided that it is possible to refold the desired peptide to the active protein form in vitro by some process (see, for example, U.S. Pat. No. 4,997,916). However, there are proteins for which in vitro refolding is highly inefficient.
Several factors appear to contribute to the formation of inclusion bodies. The most important of these are the rapid synthesis of a single protein following induction of gene expression in the fermentation process, and the temperature at which fermentation takes place. At high temperatures, refolding of the individual molecule is slowed down. The presence of a high concentration of incompletely folded proteins leads to protein aggregation due to the intermolecular interactions of hydrophobic domains. In production of the native protein, these hydrophobic domains play an important role in the folding of the protein into a fully active form.
At present, there is no known way to direct the production of proteins in their native form during the fermentation process. For some proteins (such as hGH and bGH) it is possible to disaggregate and refold the desired protein, while for others there is no known refolding process. However, it is known that chaperonin protein complexes can act to facilitate disaggregation in vivo. This process can also be carried out on a small scale in biochemical laboratories.
It has been shown that proteins expressed in E. coli at low temperatures have an increased solubility (Schein et al., Bio/Technoloqy (1988) 6:291; Schein, Bio/Technology (1989) 7:1141). For example, when interferon (IFN-.alpha.2) was expressed in a bacterial culture at 37.degree. C. 95% of the protein was found in inclusion bodies. In contrast, only 27% of the protein was insoluble when the same culture was grown at 30.degree. C. Similar results were obtained for IfN-.gamma., and Shirano et al. (FEBS (1990) 1:128) demonstrated the low-level expression of soluble lipoxygenease in E. coli at about 15.degree. C.
Accordingly, there remains a need for vectors which allow for the isolation of biologically active eukaryotic protein from bacterial cells. It is an object of the present invention to provide such vectors, as well as methods for the production and isolation of eukaryotic proteins from bacterial cells in a biologically active form. It is another object of the present invention to provide host-vector systems for the production of a desired protein in native form.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.